1. Field of the Invention
The present invention relates to new ligands for nicotinic acetylcholine receptors (nAChRs), particularly radioligands and fluorimetric ligands based on methyllycaconitine (MLA).
2. Discussion of the Background
Neuronal nicotine acetylcholine receptors (nAChRs) represent a major neurotransmitter receptor superfamily responsible for excitatory neurotransmission (Lindstrom et al. Ann. N. Y. acad Sci., 1995, 757, 100-116). The xcex14xcex2n and xcex17 nAChR are the major receptors in the human brain. These receptors are of great interest since they appear to play a critical role in tobacco dependence and neurodegenerative disease. In particular, the nAChRs have been targeted for the development of drugs for cognitive function, Parkinson""s disease, analgesia, inflammatory bowel disorder, schizophrenia, anxiety, depression, Tourette""s syndrome and smoking cessation. For example, the addictive nature of cigarette smoking can be attributed to the reinforcing properties of nicotine (Corrigall et al. Psychopharmacology, 1992, 107, 285-289), and nicotine, which binds to nAChRs with high affinity, has been utilized in various smoking cessation therapeutics (Balfour et al. Pharmacol. Ther., 1996, 72, 51-81). In post-mortem autoradiographic studies on Alzheimer""s disease tissue, several groups have consistently revealed significant reduction of nAChRs in comparison to controls (Whitehouse et al. Brain Res., 1986, 371, 146-151; Nordberg et al. Neurosci. Lett. 1986, 72, 115-119; London et al. Neurochem. Res., 1989, 14, 745-750). In addition, nicotine appears to improve cognitive functions (Lippielo Alzheimer""s Disease. Therapeutic Strategies, 1994, E. Giacobini and R. Bekcer, Ed., Boston, Birkhauser, 186-190). These results have prompted the pharmaceutical industry to explore the development of safe and effective nAChR-based therapeutic agents for treatment of Alzheimer""s disease (Brioni et al. Adv. Pharmacol., 1997, 37, 153-214).
Over the past few years considerable effort has been directed toward the identification and characterization of radioligands for nicotinic acetylcholine receptors (nAChRs) (Holladay et al, J. Med. Chem. 1997, 40, 4169-4194). Two major classes of nicotinic receptors have been identified in rat and human brain based on whether they demonstrate high affinity binding for either [3H]nicotine or [125I]xcex1-bungarotoxin ([125I]xcex1-BGT) (Marks et al, Mol. Pharmacol. 1982, 22, 554-564. Heteromeric receptors composed of xcex1 and xcex2 subunits bind [3H]nicotine with high affinity. The xcex14xcex22 receptor is the most common subtype comprising almost 90% of rat brain nAChRs (Lindstrom et al, Ciba Found Symp. 1990, 152, 23-52). Receptors with high affinity for [125I]a-BGT contain only the xcex17 subunit (Clarke et al, J. Neurosci. 1985, 5, 1307-1315; Seguela et al, J. Neurosci. 1993, 13, 596-604) and display a regional distribution distinct from the xcex1xcex2 heteromeric receptors (Marks et al, Mol. Pharmacol. 1982, 22, 554-564; Marks et al, Mol. Pharmacol. 1986, 30, 427-436). Several new tritium and iodine-125 ligands have been developed for studying the pharmacological properties of xcex14xcex22 nAChRs (Houghtling et al, Mol. Pharmacol. 1995, 48, 280-287; Davila-Garcia et al, J. Pharmacol Exp. Ther. 1997, 282, 445-45 1; Horti et al, Nucl. Med. Biol. 1999, 26, 175-182; Musachio et al, Synapse 1997, 26, 392-399; Musachio et al, Life Sci. 1998, 62, PL 351-357; Scheffel et al, NeuroReport 1995, 6, 2483-2488.). In addition, several carbon-11, fluorine-18, and iodine-123 positron emission tomography (PET) and single-photon emission computed tomography (SPECT) tracers have been developed for in vivo imaging of xcex14xcex22 nAChRs (Horti et al, Nucl. Med. Biol. 1999, 26, 175-182; Musachio et al, Synapse 1997, 26,392-399; Musachio et al, Life Sci. 1998, 62, PL 351-357; Ding et al, Synapse 1996, 24, 403-407; Ding et al, Mapping nicotinic acetylcholine receptors with PET, Society for Neuroscience, Washington, D.C., 1996, Abstract 22, 269; Horti et al, J. Labelled Compd. Radiopharm. 1996, 38, 355-365; Ding et al, Nucl. Med. Biol. 1999, 26, 139-148; Gatley et al, Nucl. Med. Biol. 1998, 25, 449-454; Ding et al, J. Label Compds. Radiopharm. 1997, 39, 827-832; Liang et al, J. Med. Chem. 1997, 40, 2293-2295; Loc""h et al, J. Labelled Compd. Radiopharm. 1997, 40, 519-521; Patt et al, Nucl. Med. Biol. 1999, 26, 165-173; Dolle et al, J. Med. Chem. 1999, 42, 2251-2259; Dolci et al, Bioorg. Med. Chem. 1999, 7, 467-479; Horti et al, J. Labelled Comp. Radiopharm. 1998, 41, 309-318; Horti et al, J. Med. Chem. 1998, 41, 4199-4206; Horti et al, Nucl. Med. Biol. 1998, 25, 599-603). At present, [125I]-xcex1-BGT is the only iodine-labeled radioligand specific for the xcex17 nAChR. xcex1-BGT is a 7800-8000 kD 74 amino acid polypeptide isolated from snake venom, Bungarus multicinctus (Mebs et al, Became. Biophys. Res. Commun. 1971, 44, 711-716.) The radioligand has the disadvantage of big nonspecific binding in filtration-based assays. Moreover, xcex1-BGT does not cross the blood-brain barrier limiting its use for imaging studies for the xcex17 nAChR.
Neuronal [125I]xcex1-BGT binding sites, a subtype of nicotinic receptors, are altered in a number of CNS disorders such as schizophrenia and Parkinson""s disease (Freedman et al. Proc. Natl. Acad. Sci. USA, 1997, 94, 587-592). A good correlation has been noted between the distribution of xcex17 mRNA subunits and that of the high affinity binding sites for xcex1-BGT in rodent brain (Clarke et al. J. Neurosci., 1985, 5, 1307-1315; Seguela et al. J. Neurosci., 1993, 13, 596-604). However, potent and selective agonists and antagonists at the xcex17 nicotinic receptor subtype are lacking. Methyllycaconitine (MLA), a natural product isolated from the seeds of Delphinium brownii, has high affinity to neuronal [125I]xcex1-BGT binding sites (Ki=4 nM), in contrast to its much weaker interactions with the xcex1-bungarotoxin-sensitive nicotinic receptor subtype present on the neuromuscular junction and with other nicotinic receptor subtypes labeled by [3H]nicotine. MLA blocked the activation of xcex17 receptor subtype expressed in oocytes with an IC50 in the picomolar range (Palma et al. J. Physiol., 1996, 491, 151-161). The selectivity of MLA towards the brain xcex1-bungarotoxin-sensitive receptor subtype, i.e. xcex17, makes this agent very useful for studying the properties of this subtype in vitro. In contrast to xcex1-BGT, MLA is a relatively small reversible binding compound. In addition, Turek et al (Turek et al. J. Neurosci. Meth. 1995. 61, 113-118) showed that peripherally administered MLA crosses the blood-brain barrier and may, therefore, be a useful tool to further probe the CNS functions of the xcex17 nicotinic receptor subunit in vivo.
In general, imaging drug and neurotransmitter receptors by PET or SPECT is very useful. For example, dopamine transporters can be imaged, and this procedure shows great promise as a diagnostic approach for Parkinson""s disease (Kuhar et al. Neurotransmitter Transporters: Structure and Function, 1997, M. E. A. Reith, Ed., Totowa, N.J., Human Press, Publishers, 297-313; Innis et al. Proc. Natl. Acad Sci. USA, 1993, 90, 11965-11969; Frost et al. Ann. Neurol., 1993, 34, 423-431) as a method to determine the doses of therapeutic drugs needed to achieve significant receptor occupancy and, therefore, therapeutic benefit (Scheffel et al. Synapse, 1994, 16, 263-268) and as a method to reflect the level of neurotransmitter present in the synapse and the activity of central cholinergic systems (Volkow et al. Synapse, 1994, 16, 255-262).
Tomographic imaging studies of central nAChRs in living subjects have been hampered by the absence of radiotracers that possess favorable in vivo properties. Although [C-11](xe2x88x92)nicotine has been utilized to study nAChRs in humans, its high nonspecific binding and flow-dependent tissue retention make it less than ideal as an in vivo probe (Nyback et al. Psychopharmacology (Berl.), 1994, 115, 31-36). Research efforts, therefore, have focused on development of new nAChR radioligands including [F-18] and [123I]-labeled analogs of the potent nAChR agonist epibatidine. These radiolabeled epibatidine derivatives have successfully imaged xcex14xcex22 nAChRs with high specificity in non-human primate (Ding et al. Synapse, 1996, 24, 403-407; Musachio et al. Synapse, 1997, 26, 392-399; Villemagne et al. J. Nucl. Med, 1997, 38, 1737-1741). No iodine-radiolabeled ligands are available for imaging the xcex17 nAChR.
Accordingly, one object of the present invention is to provide a new ligand for nAChRs that provides high specificity binding.
A further object of the present invention is to provide radioligands based on MLA.
Another object of the present invention is to provide fluorimetric ligands based on MLA.
Another object of the present invention is to provide an assay for nAChR activity using the ligand of the present invention for detection and quantitation.
These and other objects of the present invention have been satisfied by the discovery of ligands for nAChRs having the structure: 
where R1 is a detectable marker group, preferably a radioisotope or a fluorimetric marker group, and its use in imaging and assays for measuring nAChR activity.
The present invention relates to ligands for nAChRs based on methyllycaconitine having the formula (I): 
where R1 is a detectable marker group. The detectable marker group can be any group that can be sensitively detected using conventional methods. Preferably, R1 is a radioisotope or a fluorimetric marker group. The present ligands have high specificity for the xcex17 nAChR sites and provide diagnostic imaging agents for detection of Alzheimer""s disease and other CNS disorders and determination of dosing levels for CNS disorders. In addition, the ligands can be used in high throughput assays using rat brain homogenates for detection of xcex17 nAChR compounds synthesized by combinatorial methods.
The ligand of the present invention is preferably a radioligand, as these can be used in both in vitro assays and in vivo imaging. Suitable radioisotopes for use as R1 include 125I, 123I, 124I, 120I, 11C (as part of a C1-C4-alkyl group), and 18F, with the Iodine radioisotopes being preferred, most preferably 125I or 123I. The ability of MLA compounds to cross the blood-brain barrier makes the present ligands particularly useful for in vivo imaging of the living human brain. Most preferably, the present ligand is 125I-MLA (R1=125I) having the structure [125I]iodo-MLA shown in Scheme I. For SPECT imaging studies, the [123I]iodo-MLA compound, having the 123I substitution in the same location as the 125I, is preferred. This compound is readily prepared by the same method shown in Scheme I, using sodium [123I] iodide in place of sodium [125I]iodide. 
For in vitro assays, such as combinatorial assays, the ligand can use any readily detectable marker group, preferably radioisotopes, or fluorimetric marker groups as R1. Suitable radioisotopes include those noted above. Fluorimetric marker groups must be capable of generating a fluorescent signal, but must not interfere with the binding specificity of the MLA molecule with xcex17 nAChRs. Suitable fluorimetric marker groups include those having the structures a-m, below. 
These groups are well known fluorimetric markers that can be provided on the MLA molecule using conventional synthetic techniques, particularly from the I-MLA or trimethylstannyl-MLA compound of Scheme I. Also, the fluorimetric markers can be provided by preparation of the nitro-MLA compound (having xe2x80x94NO2 in place of I), followed by reduction to amino and coupling of the fluorimetric marker group to the MLA structure. The methods for these reactions are well known in the art. Most preferably, the fluorimetric marker group is a group having a structure selected from the group consisting of structures a-h, with the most preferred groups being those with longer linking chains (structures b-h).
The most preferred embodiment, [125I]iodomethyllycaconitine ([125I]iodo-MLA), can be synthesized by the route outlined in the following Scheme I. MLA is isolated from Delphinium elatum (Pacific giant) seeds according to the procedure developed by Pelletier and co-workers (Pelletier et al, Tetrahedron 1989, 45, 1887-1892). Alkaline hydrolysis of MLA using 5% potassium hydroxide in ethanol gives the ester-free lycaconitine (2) (Pelletier et al, J. Nat. Prod. 1980, 43, 395-406). Treatment of 5-iodoanthranilic acid (3) with (S)-methylsuccinic anhydride (4) provides a mixture of the iodomethyllycaconitinic acids (5 and 6). The 1H-NMR spectrum of the acids show two equal doublets at 1.29 and 1.26 ppm (J=7.1 Hz) for the xe2x80x94CHxe2x80x94CH3 groups which suggests that the acids are a 1:1 mixture of 5 and 6. The mixture of acids is refluxed under a Dean Stark tube in toluene containing triethylamine for 24 h to yield (S)-2-(methylsuccinimido)-5-iodobenzoic acid (7). The structure and single isomer nature of 7 is established by the 1H NMR spectrum which shows only one doublet at 1.37 ppm (J=6.8 Hz) for the CHxe2x80x94CH3 group. The acid 7 is coupled to the primary alcohol group of lycoctonine in the presence of p-toluenesulfonyl chloride and pyridine to give iodo-MLA. Refluxing iodo-MLA with hexamethyldistannane in toluene in the presence of palladium-tetrakis-triphenylphosphine provides trimethylstannyl-MLA which is the precursor needed to prepare [125I]iodo-MLA. This trimethylstannyl-MLA can also be used as the starting material for a variety of reactions to generate the various ligands of the present invention, including radio-ligands and fluorimetric ligands, using conventional chemical reactions well known to those of skill in the art.
The present invention also provides compounds that are precursors to the ligands of the present invention. In particular, these precursors have R1 being a group that is readily converted into the desired detectable marker group. Preferably, R1 in the precursor compounds is tri-hydrocarbyl-silyl or tri-hydrocarbyl-stannyl, preferably the trimethylsilyl and trimethylstannyl compounds. Most prefereably, the precursor compound is a trimethylstannyl-MLA compound. In a typical preparation of a radioligand and use in imaging, the person doing the imaging study will purchase the trimethylstannyl-MLA compound and convert it to the radioligand immediately prior to use, particularly if the radioligand has a relatively short half-life. This avoids degradation of the radioligand prior to use.
A sample of trimethylstannyl-MLA used for radio-iodination was purified by HPLC to eliminate any of iodo-MLA from the precursor, as the presence of unlabeled iodo-MLA would reduce the specific activity of the final radiolabeled product. HPLC analysis showed that the contamination of iodo-MLA in the trimethylstannyl-MLA precursor was less than 0.027%. Since the ratio of trimethylstannyl-MLA/Na125I was 17, the effect on specific activity of the labeled product was less than 0.46% (0.027% 17), which was within normal experimental error. The radio-iodination (radio-iododestannylation) process of the precursor was completed within one minute at room temperature using chloramine-T as oxidant. The total radiochemical yield of [125I]iodo-MLA after HPLC purification was 74%.
The pharmacological profile of [125]iodo-MLA, based on the competition binding studies of compounds binding to the xcex14xcex22, and non-competitive nAChRs, is consistent with this radioligand being specific for the xcex17 nAChR subtype. Again, this is in agreement with the pharmacological profile of MLA (Alkondon et al, Mol. Pharmacol. 1992, 41, 802-808; Wonnacott et al, Methods in Neurosciences 1993; Vol. 12, pp 263-275) and [3H]MLA (Davies et al, Neuropharmacology 1999, 38, 679-690).
The high specificity binding of the present ligands to xcex17 nAChRs make it preferable to [125I]xcex1-BGT in the study of this nAChR subtype. Moreover, its high specific activity makes it suitable for use in high throughput screening assays aimed at identifying nAChR subtype-specific ligands through competitions binding to determine candidates having higher specific affinity to the nAChRs. Since the addition of iodine-125 to MLA does not alter its specificity for the xcex17 nAChR, an iodine-123 labeled MLA would be a useful ligand for imaging this nAChR subtype in vivo. Finding nAChR subtype specific ligands that are useful as an imaging agent is particularly relevant since reduced numbers of nAChRs have been observed in Parkinson""s and Alzheimer""s diseases and in schizophrenia (Brioni et al, Adv. Pharmacol. 1997, 37, 153-214).
Imaging techniques are well known in the art. Because radiolabeled iodo-MLA binds preferentially to xcex17 nAChRs in vivo, it can be used as an imaging agent for both PET (Positron Emission Tomography) and SPECT scanning. PET scanning preferably uses the carbon-11 labeled form of the drug, while SPECT scanning preferably uses the I-123 labeled form of the drug. It can be used in the following ways.
A. To examine the density and distrubition of certain xcex17 nAChRs in various parts of the body, including the brain.
B. To compare these densities in normal and disease states and use observed changes that can be associated with diseases as indicators diagnostic of disease states. The invention may also be employed to determine progression of the disease and/or prognosis as to various treatment regimens.
A brief description of an imaging procedure is as follows:
Tracer quantities (25-100 xcexcg) of the radioactive iodine labeled ligand is injected intravenously into subjects positioned in a SPECT scanner. After injection of the compound, the scanner is turned on to begin to collect data. The ligand preferentially localizes to xcex17 nAChRs over about one hour, with the best localization occurring at about 30-50 minutes. The amount of compound bound will reflect the density of xcex17 nAChRs. The target of these experiments will be the hippocampus thalamus/hypothalamus where the xcex17 nAChRs are concentrated. Disease states such as Parkinson""s and Alzheimer""s diseases will show a reduction in xcex17 nAChRs density.
The present invention also provides an injectable composition suitable for use in imaging studies comprising a compound of Formula I where R1 is a marker group detectable in vivo and a pharmacologically acceptable carrier. The injectable composition should contain an amount of ligand sufficient to administer up to 30 xcexcg, preferably up to 25 xcexcg, of ligand in a single injection. Suitable pharmacologically acceptable carriers include any conventional carriers used for injecting a patient particularly in imaging studies. Preferably the carrier is water.
The present invention also relates to a kit for imaging, comprising a compound of formula I where R1 is a group that can be converted into the marker group detectable in vivo (such as trimethylstannyl), the reagents for performing the conversion into the marker group detectable in vivo (such as those shown in Scheme I for conversion of trimethylstannyl into the [125I]-MLA compound) and a pharmacologically acceptable carrier.